**2.1. Polystyrene NPs as a VADS**

**1. Introduction**

28 Immunization - Vaccine Adjuvant Delivery System and Strategies

cines or immunotherapy [2].

Vaccines have saved countless human lives from lethal infections since the modern use of vaccinia against smallpox by British physician Edward Jenner in the late eighteenth century and are today playing more and more crucial roles in fighting life-threatening diseases, of which, due to great advances made in branches of the field and related fields such as immunology and biotechnology, the scope has enormously expanded, ranging from the earlier aim of microorganism infections to the novel targets of autoimmune disorders, allergic reactions, and even malicious cancers [1]. However, the list of infectious diseases for which vaccines are urgently needed but do not yet exist is till long and in particular many pathogens such as HIV (human immunodeficiency virus), HSV (herpes simplex virus), and HCV (hepatitis C virus), showing on their surface elusive or ever changing immunogenic features to continuously dismantle a variety of potential arsenals; let alone numerous malignant tumors, autoimmune disorders such as multiple sclerosis, diabetes, and rheumatoid arthritis, which are all aggressively threatening human health and life, posing a big challenge to developing effective vac-

Vaccines function against diseases through stimulating the host immune system with the antigenic components featured by pathogens or neoplasms to establish the antigen-specific immunity which is able to clear the abnormalities bearing the identical antigens. Vaccines developed for handling infectious diseases are mostly manufactured using the live attenuated or killed whole microbes, which have a high potency in triggering immune system but are argued to be associated with possible reversion to virulence due to mutation of administered strains, as evidenced by gene sequencing in vaccinated sufferers, thus causing safety concern. As many mechanisms underlying immunoresponses are revealed and great achievements are made in relevant fields, subunit vaccines, which are formulated with defined components including Ags to induce immunoresponses accurately targeting the matched objects thus causing few safety concerns, are now more and more employed to fight not only infectious pathogens but also other illnesses, providing, in particular, outstanding ways to overcome the previously intractable diseases, such as cancer [3]. Compared to conventional whole microbe vaccines, subunit vaccines possess many distinct properties which are beneficial for clinical applications, summarily including high safety profile, production needing no dangerous microorganisms, no redundant components to cause allergic or autoimmune responses, diverse usage including anticancer, and high capacity for several peptide epitopes targeting

However, subunit vaccines are often weak immunostimulatory products, due to lack of such components as the pathogen-associated molecular patterns (PAMPs), which are expressed on a microbe surfaces and are able to activate the pattern recognition receptors (PRRs), such as the TLRs (toll-like receptors), NOD-like receptors (the nucleotide-binding oligomerization domain-like receptors), RIG-I-like receptors (retinoic-acid-inducible gene-I-like receptor), and C-type lectin receptors, thus facilitating host immunoresponses [5]. As such, subunit vaccines are often formulated with an adjuvant, which is a nonspecific immunopotentiating substance able to elevate, either in advance or simultaneously with the vaccine Ags, the immune

different stages in the life cycle or subtypes of a pathogen [4].

Although polymers suitable for constructing VADSs are usually thought to be biodegradable one since they cause no size-limited excretion and associated toxicity concern, certain nonbiodegradable materials possessing certain specific properties, such as chemical inertness and ease for fabricating stable NPs, are also the preferred candidates by researchers for engineering the kind of NPs with an accurate size and special shape, so as to be employed reliably to investigate these physical properties on the immune system and immune responses. For example, Plebanski and coworkers using nonbiodegradable polystyrene NPs performed studies on VADS and showed that polystyrene NPs loaded with OVA epitopes induced different immune responses in a size-dependent manner needing no additional adjuvant, and that among different particles with a size ranging from 20 to 2000 nm, 40 nm NPs induced the strongest cellular and humoral immunity [12]. Further investigations demonstrated that covalent linkage of peptide to NPs is a requirement for eliciting immunization efficacy and also proved that 40-nm-sized NPs serve as a VADS owing to their preferential uptake by APCs and their ability to traffic to lymph nodes to induce strong immune responses compared to their larger counterparts [4, 13].

protect against different virus genotypes, high potency to trigger immune responses providing an option to conquering intractable pathogens such as HIV and HCV (hepatitis C virus), high thermostability possibly requiring no integral cold chain to keep viability favoring global vaccination, and high manufacturing efficiency in large scale while at low cost offering a strategy to handle the emergency arising from infectious diseases, such as Ebola outbreak and epidemic. For example, HCV infection is still a significant public health problem, though it has been partially addressed with the advent of directly acting antiviral agents (DAAs), which represent a major advance toward controlling HCV but confer little protection against reinfection [18]. Presently, around 71 million people in the world are living with chronic HCV infection, and each year nearly half a million of them will die of HCV infection or its complications, rendering it urgent to develop an effective vaccine capable of eradicating HCV, which may well be produced using the VLP-based VADS, in reference to HBV vaccines. Recently, a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine was successfully engineered by researchers using scale-up production methods of Huh7 cell factories containing a recombinant adenoviral expression system representing each HCV genotype, followed by cell lysing and purification with iodixanol ultracentrifugation and stirred cell ultrafiltration [19]. When given subcutaneously to mice, whether in the presence of an adjuvant (system) or not, the quadrivalent vaccine consistently induced production of Ab and nAb (neutralizing Ab) together with robust T and B cell responses for eliciting broad humoral and cellular immunity, indicating the VLP-based VADS a useful tool which may be employed for the production of an effective HCV vaccine [20].

Polymeric Nanoparticles Engineered as a Vaccine Adjuvant-Delivery System

http://dx.doi.org/10.5772/intechopen.81084

31

Globally, HIV continues to be a major public health issue and also claims approximately 1 million lives each year, although total people living with HIV have the opportunity to receive antiretroviral drugs (ARVs), which may effectively control the virus from transmission and causing illness but are also found to be undermined in efficacy by pathogens that had evolved with drug resistance [21]. Still, a highly effective vaccine is believed to be the ultimate weapon able to erase HIV and associated disease AIDS (acquired immune deficiency syndrome), though at present, there are no such a product in markets and, moreover, many products developed in previous years bearing such expectations failed to show clinical efficacy in fighting this rapidly mutating pathogen, including especially the big trial, known as STEP, which was halted in 2007 after the vaccine was found to increase the risk, instead of prophylaxis, of HIV infection [22]. Nevertheless, scientists are getting closer than ever to developing such an effective product, as evidenced by a large-scale clinical study conducted in 2009 in Thailand (called RV144) showing that immunization with a combination of two HIV vaccines prevented about 31% of new infections through a prime-boost combination regime [23], which comprises four priming intramuscular injections of ALVAC-HIV, which is a recombinant canarypox vaccine express HIV-1 Gag, Pro and gp120-gp41, plus two boosting intramuscular injections of AIDSVAX® B/E, which is an alum-adjuvanted bivalent HIV-1 gp120 vaccine of subtypes E and B [24]. Though the low prevention rate of vaccination with ALVAC/AIDSVAX combination excludes the products from approval for clinical prophylaxis of HIV, the moderate effects displayed in human trial not only provoked scientists to make deep explorations on the causes for the failures, but also presented researchers a great encouragement to commit further efforts to developing efficacious HIV vaccines. Subsequently, based on the virion features which are more and more clearly elaborated in structure and function, scientists set out to handle the obstacles identified to the development of a preventive HIV vaccine from several aspects, such as accurately targeting the conserved antigenic proteins, seeking Ags able

Notably, Schöttler et al*.* recently reported the counterintuitive research results on cellular uptake of NPs with PEGylation (modification with polyethylene glycol), which is gold standard in removal of immune clearance of in vivo NPs through the mechanism of reducing nonspecific cellular uptake of nanocarriers [14]. The researchers documented that polystyrene NPs, which had been modified with PEG or poly(ethyl ethylene phosphate) (PEEP), only had been exposed to plasma proteins, could exhibited a lowered cellular uptake by macrophages (RAW264.7 cells), whereas those not exposed to plasma proteins showed high nonspecific uptake. Further mass spectrometric analysis revealed that the plasma-exposed nanocarriers formed a protein corona which was identified to contain just an abundance of clusterin proteins (known as apolipoprotein J) and to be the decisive factor controlling lowered nonspecific cellular uptake of the PEGylated or PEEPylated polystyrene NPs, and to contrast, the classic conception that PEGylated NPs free of immune clearance is resulted from avoidance of protein adsorption. These outcomes indicated that PEG as well as PEEP can affect the composition of protein adsorption by polystyrene NPs, and that the presence of certain type proteins may be just a prerequisite in preventing nonspecific cellular uptake of NPs, defying the conventional belief that PEGylation reduces protein adsorption thereby conferring a stealth effect [15].
